Intein介导的重组昆虫毒素BmK IT在大肠杆菌中的可溶性表达、纯化及活性分析

为获得重组蝎昆虫毒素BmKIT,通过PCR方法在BmKIT基因的3′端融合了编码6个组氨酸残基的核苷酸序列,将其插入原核表达载体pTWIN1的内含肽Ssp DnaB Intein基因下游的多克隆位点(MCS)。将获得的表达质粒转化大肠杆菌BL21(DE3)中,用IPTG诱导融合蛋白表达。用Ni-NTA亲和层析柱从菌体裂解液中纯化了CBD-Intein-BmK IThis6融合蛋白,并在柱上诱导Intein自剪切,成功去除融合子CBD-Intein。通过Superdex75凝胶过滤层析获得了纯度达95%以上的BmK IThis6蛋白,该蛋白不仅具有正确的二级结构而且有生物活性。     

Expression and purification of the BmK M1 neurotoxin from the scorpion Buthus martensii Karsch

The gene encoding a neurotoxin (BmK M1) from the scorpion Buthus martensii Karsch was expressed in Saccharomyces cerevisiae at a high level with the alcohol dehydrogenase promoter. SDS-PAGE of the culture confirmed expression and showed secretion into medium from yeast. Recombinant BmK M1 was purified rapidly and efficiently by ion exchange and gel filtration chromatography to homogeneity, produced a single band on tricine-SDS-PAGE, and processed the homologous N-terminus. Amino acid analysis and N-terminal sequencing demonstrated that the recombinant toxin was processed correctly from the alpha-mating factor leader sequence and was chemically identical to the native form. The expressed recombinant BmK M1 was toxic for mice, which indicated that it was biologically active. Quantitative estimation showed that recombinant BmK M1 had an LD(50) similar to that of the native toxin.     

Cloning,co-expression with an amidating enzyme,and activity of the scorpion toxin BmK ITa1 cDNA in insect cells

BmK ITa1 is an insect-specific neurotoxin from the Chinese scorpion Buthus martensi Karsch (Bmk). We succeeded in obtaining biologically active recombinant BmK ITa1 protein by simultaneous expression in insect cells of BmK ITa1 cDNA with an amidating enzyme expressed by the rat peptidylglycine α-amidating monooxygenase (PAM) gene. We investigated the insecticidal efficacy of recombinant BmK ITa1/W (without coexpression of PAM), and of BmK ITa1/A (with coexpression of PAM) in 5th instar Bombyx mori, by injecting these recombinant toxins into larvae. The lethal time for 50% of larvae (LT₅₀) was 9 h for BmK ITa1/A and 17 h for BmK ITa1/W. At 19 h after injection all of the larvae exposed to BmK ITa1/A had been killed, whereas only half of the larvae exposed to BmK ITa1/W had been killed. These results show that the simultaneous expression of an amidating enzyme can result in apparently higher insecticidal activity of BmK ITa1.     

Expression and Purification of the BmK M1 Neurotoxin from the Scorpion Buthus martensii Karsch

The gene encoding a neurotoxin (BmK M1) from the scorpion Buthus martensii Karsch was expressed in Saccharomyces cerevisiae at a high level with the alcohol dehydrogenase promoter. SDS–PAGE of the culture confirmed expression and showed secretion into medium from yeast. Recombinant BmK M1 was purified rapidly and efficiently by ion exchange and gel filtration chromatography to homogeneity, produced a single band on tricine–SDS–PAGE, and processed the homologous N-terminus. Amino acid analysis and N-terminal sequencing demonstrated that the recombinant toxin was processed correctly from the α-mating factor leader sequence and was chemically identical to the native form. The expressed recombinant BmK M1 was toxic for mice, which indicated that it was biologically active. Quantitative estimation showed that recombinant BmK M1 had an LD₅₀ similar to that of the native toxin.     

High-level expression and purification of an analgesic peptide from Buthus martensii Karch

BmK AngM1, a scorpion peptide isolated from Buthus martensii Karch was reported to exhibit potential analgesic effect. But the relative low content of this toxin in crude venom limits its further characterization. In this study, we constructed an expression vector and transformed into E.coli. The BmK AngM1 was expressed as a fusion protein in the soluble fraction and was purified by Nickel affinity chromatography. Subsequently, the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC. We purified 25 mg recombinant BmK AngM1 (rBmK AngM1) from 1 L bacterial culture. The molecular weight of rBmK AngM1 determined by ESI-MS was 7240.4 Da which was the expected size for correctly processed. Analgesic bioassay studies of rBmK AngM1 exhibited its potential analgesic effect comparable to that of the natural BmK AngM1 peptide.     

东亚钳蝎神经毒素基因BmK IT3的克隆及其在大肠杆菌中的表达

东亚钳蝎毒素基因ＢｍＫＩＴ3 编码是由 6 5个氨基酸残基组成的多肽物质。该类毒素为专一性作用于昆虫的抑制型神经毒素 ,它已被广泛用于研究离子通道作用机理[1 ] ;同时 ,它是研究蛋白质结构和功能的极好模型 ,是研究神经药理学的理想工具 ,将具有药理活性和昆虫毒性的基因导入细胞或动植物体内具有十分重要的应用价值。它对昆虫作用的专一性很高 ,对哺乳动物无害或毒性很小 ,可作为一种安全、有效的生物杀虫剂[2 ,3 ] 。我们的研究是对该基因密码子进行优化 ,采用化学合成的方法合成了适于在昆虫中表达的ＢｍＫＩＴ3 的两条长的引物 ,通过…     

Effect of location of the His-tag on the production of soluble and functional Buthus martensii Karsch insect toxin

The low yield and poor folding efficiency in vivo of soluble and active recombinant cysteine-rich proteins expressed in Escherichia coli are a major challenge for large-scale protein production and purification. Expression vectors containing Buthus martensii Karsch insect toxin (BmK IT) fused to the C terminus of the intein Ssp DnaB were constructed in an attempt to overcome this problem. Following purification and intein self-cleavage, the fusion protein His(6)-intein-IT produced insoluble BmK IT, while intein-IT-His(6) generated soluble and properly folded BmK IT. This result indicated that the positioning of the His(6) tag has a key role in the production of soluble and functional BmK IT.     

Expression and purification of the BmK Mm2 neurotoxin from the scorpion Buthus martensii Karsch and its biological activity test

The gene encoding neurotoxin (BmK Mm2) from the scorpion Buthus martensii Karsch was expressed in Escherichia coli BL21 (DE3) at a level of 1.6 mg/L using expression plasmid pExSecI system. SDS-PAGE analysis of the cell lysate confirmed that gene BmK Mm2 was expressed in soluble form and the expressed production was secreted into Luria-Bertani (LB) culture medium from Escherichia coli. According to the characters of pExSecI expression system, the IgG binding domain-ZZ of Protein A is fused to the N-terminal of BmK Mm2. Recombinant BmK Mm2 (ZZ-BmK Mm2, pI 6.81, 22.007 kDa) was purified rapidly and efficiently by IgG-Sepharose 6 Fast Flow and Superdex-75 gel filtration chromatography, produced a single band on SDS-PAGE. Western blot analysis demonstrated that this protein was recombinant BmK Mm2. The results of MTT assay, morphological observation of nucleus and single cell gel electrophoresis showed that the expressed recombinant BmK Mm2 was toxic for glial cells of mice, which indicate that it has biological activity.     

Expression, renaturation and functional analysis of an excitatory insect-specific toxin from scorpion Buthus martensii Karsch

The cDNA of BmK IT-AP, an excitatory insect toxin from the scorpion Buthus martensi Karsch that has an analgesic effect on mammalian cells, was expressed in E. coli in the form of an inclusion body. Following denaturation and reduction, the recombinant protein was renatured and purified by liquid chromatography. The authenticity of the recombinant product was confirmed by bioassay and its electrophysiological effect on insect sodium channel.     

Cloning and characterization of BmK86, a novel K+ -channel blocker from scorpion venom

Scorpion venom represents a tremendous hitherto unexplored resource for understanding ion channels. BmK86 is a novel K+ -channel toxin gene isolated from a cDNA library of Mesobuthus martensii Karsch, which encodes a signal peptide of 22 amino acid residues and a mature toxin of 35 residues with three disulfide bridges. The genomic sequence of BmK86 consists of two exons disrupted by an intron of 72 bp. Comparison with the other scorpion toxins BmK86 shows low sequence similarity. The GST-BmK86 fusion protein was successfully expressed in Escherichia coli. The fusion protein was cleaved by enterokinase and the recombinant BmK86 was purified by HPLC. Using whole-cell patch-clamp recording, the recombinant BmK86 was found to inhibit the potassium current of mKv1.3 channel expressed in COS7 cells. These results indicated that BmK86 belongs to a representative member of a novel subfamily of alpha-KTxs. The systematic number assigned to BmK86 is alpha-KTx26.1.     

Soluble expression, purification and the role of C-terminal glycine residues in scorpion toxin BmK AGP-SYPU2

The existence of glycine residues in long-chain scorpion toxins has been well documented. However, their role as analgesics has not been evaluated. To address this issue, we investigated the functional role of glycines in the C-terminal end of Chinese-scorpion toxin from Buthus martensii Karsch (BmK AGP-SYPU2) using site-directed mutagenesis and analgesic activity assays. Recombinant BmK AGP-SYPU2 and its mutants were efficiently expressed in E. coli and purified to homogeneity using immobilized metal ion affinity chromatography (IMAC) and cation exchange chromatography. The mouse-twisting test was used to detect the analgesic activity of BmK AGP-SYPU2 and its mutants. As a result, we identified glycines at the C-terminal end that, when altered, significantly affected analgesic activity. Also, Mut6566 was significantly decreased compared to BmK AGP-SYPU2. These data indicate that the glycines at the C-terminal end are important for the analgesic activity of BmK AGP-SYPU2.     

Cloning,expression, and pharmacological activity of BmK AS,an active peptide from scorpion <Emphasis Type="Italic">Buthus martensii</Emphasis> Karsch

BmK AS is a β long-chain scorpion peptide from the venom of Buthus martensii Karsch (BmK). It was efficiently expressed as a soluble and functional peptide in Escherichia coli, and purified by metal chelating chromatography. About 4.2 mg/l purified recombinant BmK AS could be obtained. The recombinant BmK AS maintained a similar analgesic activity to the natural one in both the mouse-twisting test and hot-plate procedure. It also exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. BmK AS is the first long-chain scorpion peptide reported to have antimicrobial activity, and is a valuable molecular scaffold for pharmacological research.     

Binding characteristics of BmK I, an alpha-like scorpion neurotoxic polypeptide, on cockroach nerve cord synaptosomes.

In this study, the binding characteristics of BmK I, an alpha-like neurotoxic polypeptide purified from the venom of the Chinese scorpion Buthus martensi Karsch, were investigated on rat brain and cockroach nerve cord synaptosomes. The results showed that BmK I can bind to a single class of noninteracting binding sites on cockroach nerve cord synaptosomes with medium affinity (Kd = 16.5 +/ - 4.4 nM) and low binding capacity (Bmax = 1.05 +/- 0.23 pmol/mg protein), but lacks specific binding on rat brain synaptosomes. BmK AS, BmK AS-1 (two novel sodium channel-blocking ligands), BmK IT (an excitatory insect-selective toxin) and BmK IT2 (a depressant insect-selective toxin) from the same venom were found to be capable of depressing BmK I binding in cockroach nerve cord synaptosomes, which might be attributed to either allosteric modulation of voltage-gated Na+ channels by these toxic polypeptides or partial overlapping between the receptor binding sites of BmK I and these toxins. This thus supported the notion that alpha-like scorpion neurotoxic polypeptides bind to a distinct receptor site on sodium channels, which might be similar to the binding receptor site of alpha-type insect toxins, and also related to those of BmK AS type and insect-selective scorpion toxins on insect sodium channels.     

Expression,purification and functional characterization of a recombinant scorpion venom peptide BmTXKbeta

BmTXKbeta, a scorpion toxin isolated from the Chinese scorpion Buthus martensii Karsch (BmK), was expressed as a GST fusion protein in BL21 (DE3) strain. The recombinant GST-BmTXKbeta protein was purified by affinity chromatography. When treated with enterokinase, the GST-BmTXKbeta fusion protein released an approximate 6.5kDa protein which was the expected size for correctly processed. About 2mg purified recombinant BmTXKbeta protein (rBmTXKbeta) was produced from 1l bacterial culture, using this expression and purification system. The function of rBmTXKbeta was studied on the rabbit atrial myocyte by whole-cell patch clamp technique. The results showed that rBmTXKbeta inhibited the transient outward current (I(to)) of rabbit atrial myocyte with recovery after washout and the inhibition was concentration-dependent. The rBmTXKbeta prolonged the action potential duration of rabbit atrial myocyte in a concentration-dependent manner, whereas it did not affect the action potential amplitude.     

Expression, purification and characterization of an analgesic peptide from Buthus martensii Karsch in Pichia pastoris

BmK AngM1 is an analgesic peptide from the venom of Buthus martensii Karsch (BmK). The synthetic gene encoding BmK AngM1 was optimized on the basis of its cDNA sequence and the codon usage preference of Pichia pastoris. The codon-optimized gene was cloned into pPIC9K and then transformed into P. pastoris. SDS-PAGE and Western blot analysis showed that the recombinant BmK AngM1 (rBmK AngM1) was expressed by the addition of methanol to the medium, and its maximum production reached above 500 mg/l. The purified rBmK AngM1 could be obtained efficiently by Nickel affinity chromatography. Analgesic bioassay, by the mouse-twisting model, showed that rBmK AngM1 had evident analgesic effect with an ED₅₀ of 0.5 mg/kg.     

Production of a recombinant antibody fragment in whole insect larvae

Infection of insect cells with baculovirus expression constructs is commonly used to produce recombinant proteins that require post-translational modifications for their activity, such as mammalian proteins. However, technical restraints limit the capacity of insect cell-based culture systems to be scaled up to produce the large amounts of recombinant protein required for human pharmaceuticals. In this study, we designed an automated insect rearing system and whole insect baculovirus expression system (PERLXpress™) for the expression and purification of recombinant proteins on a large scale. As a test model, we produced a recombinant mouse anti-botulinum antibody fragment (Fab) in Trichoplusia ni larvae. A recombinant baculovirus co-expressing the Fab heavy and light chains together with N-terminal sequences from the silkworm hormone bombyxin, to direct proteins into the secretory pathway, was constructed. Fifth instar larvae were reared and infected orally with recombinant (pre- occluded) baculovirus using the automated system and harvested approximately after 4 days. The total yield of recombinant Fab was 1.1 g/kg of larvae, resulting in 127 mg of pure Fab in one production run. The Fab was purified to homogeneity using immobilized metal affinity chromatography, gel filtration, and anion exchange chromatography. The identity of the purified protein was verified by Western blots and size-exclusion chromatography. Purified recombinant Fab was used to detect botulinum toxin in ELISA experiments, demonstrating that the heavy and light chains were properly assembled and folded into functional heterodimers. We believe that this is the first demonstration of the expression of a recombinant antibody in whole insect larvae. Our results demonstrate that a baculovirus-whole larvae expression system can be used to express functionally active recombinant Fab fragments. As the PERLXpress™ system is an automated and linearly scalable technology, it represents an attractive alternative to insect cell culture for the production of large amounts of human pharmaceuticals.     

转BmK IT4基因烟草的抗虫性

The BmK IT4 gene was obtained from pBS-BmK IT4 by EcoRⅠ/KpnⅠdigestion and it was then cloned into pE3 intermediate vector. The resulting plasmid was named pE3-BmK IT4. The chimeric gene was transferred into the tobacco (Nicotiana tabacum L.) genome via Agrobacterium-mediated transformation. Forty-five regenerated kanamycin resistant plants were obtained, two individual lines showed strong toxicity to Manduca sexta (Linnaeus), Heliothis armigera (Hübner) and Leguminivora glycinivorella (Matsumura) by feeding experiments. Results from Southern blot indicated that BmK IT4 gene was transferred into tobacco genome. The mortality of M．sexta, H．armigera and L．glycinivorella larvae fed on transgenic plants was 95%-97%, 63%-70% and 65%-73%, respectively, and the growth of the surviving insects was remarkably retarded.     

人血管生成素-PE40融合蛋白基因的构建、表达及活性研究

利用 PCR扩增出人血管生成素 (h ANG)成熟肽的基因片段 .与绿脓杆菌外毒素缺失突变体PE40的基因连接后 ,克隆入 p UC1 9载体中 .测序后克隆入表达载体 p RSETB,构建成 h ANG-PE40融合基因的表达载体 .IPTG诱导 ,表达出分子量约为 58k D的 His6- ANG- PE40融合蛋白 ,占菌体总蛋白的 8% .Ni2 +- NTA树脂纯化表达蛋白 ,SDS- PAGE结果显示纯化重组蛋白为单一条带 .鸡胚绒毛尿囊膜鉴定表明重组蛋白体外能够有效地抑制血管的形成 .     

Electrophysiological characterization of BmK M1, an alpha-like toxin from Buthus martensi Karsch venom

The present study investigates the electrophysiological actions of BmK M1, an alpha-like toxin purified from the venom of the scorpion Buthus martensi Karsch, on voltage-gated Na+ channels. Using the voltage clamp technique, we assessed the BmK M1 activity on the cardiac Na+ channel (hH1) functionally expressed in Xenopus oocytes. The main actions of the toxin are a concentration-dependent slowing of the inactivation process and a hyperpolarizing shift of the steady-state inactivation. This work is the first electrophysiological characterization of BmK M1 on a cloned Na+ channel, demonstrating that this toxin belongs to the class of scorpion alpha-toxins. Our results also show that BmK M1 can be considered as a cardiotoxin.     

杆状病毒重组蛋白金鱼生长激素Ⅰ的纯化

利用放射免疫分析(RIA)证实了含金鱼生长激素ⅠcDNA的重组病毒在感染细胞96h后所表达的生长激素达到最高水平,平均每105个细胞可分泌金鱼生长激素Ⅰ最高达288.07ng;平均每克干虫可产生金鱼生长激素Ⅰ 925.3μg。从感染重组病毒的无血清细胞培养基中纯化生长激素Ⅰ,平均每毫升培养基可纯化具免疫活性纯度95%以上的金鱼生长激素Ⅰ达664.224ng。纯化后蛋白的N\|末端首20个氨基酸序列测定的结果表明其信号肽得到了准确的切割。从而保障了表达蛋白的生物活性。